Arteriovenous (AV) fistulas provide the optimal vascular access for hemodialysis in patients with end stage renal disease (ESRD), yet nearly 50% of fistulas fail soon after their surgical construction due, in part, to intimal hyperplasia of venous smooth muscle cells (SMC) that occludes the fistula lumen. Oxidative stress and reduced nitric oxide (NO) bioavailability are important contributors to the accelerated vascular disease that accompanies ESRD, and accumulating data suggest the same factors account for aggressive development of venous intimal hyperplasia. However, processes underlying intimal hyperplasia in the setting of uremia, and, specifically, in the venous outflow of hemodialysis AV fistulas, have not been delineated. The central hypothesis of this proposal is that potentially reversible oxidant-induced impairment of the inhibitory action of NO on vascular SMC migration and proliferation, present at the time of fistula creation, leads to pathologic neointimal hyperplasia and maturation failure. In this R21 application we propose proof- of-concept and feasibility studies to provide the groundwork for a more definitive investigation of this hypothesis that should lead to mechanism-based development of maturation-enhancing interventions. Our proposal has the following aims: 1) To evaluate relationships between venous SMC responsiveness to NO and fistula maturation outcomes. Using explant cultures of venous SMC, that have a dedifferentiated phenotype similar to that of cells responsible for neointimal growth in newly created fistulas, we will evaluate proliferation and migration in response to serum growth factors as well as their inhibition by NO, and compare the responses between cells derived from veins that ultimately do or do not mature successfully. We will quantify NO release, measure reactive oxygen species, determine the redox status of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and assess intracellular Ca2+. 2) To evaluate relationships between preexisting venous tissue expression of mediators of NO bioactivity and its smooth muscle functions and subsequent fistula maturation outcomes. We will use real-time quantitative PCR to evaluate expression of genes that regulate SMC function via NO pathways. These include eNOS, iNOS, guanylyl cyclase, protein kinase G, NADPH oxidase isoforms, Ca2+ channels, and SERCA. Alterations in gene expression will be confirmed by immuno-histochemistry on additional vein tissue fixed at the time of fistula creation. Our focus on tissue and cellular gene expression rather than plasma concentrations of these NO mediators is based on our expectation that local expression is more directly relevant to SMC function and maturation outcomes. 3) To determine whether impaired NO responsiveness can be restored by over- expression of antioxidant enzymes, down-regulation of oxidant producing enzymes, or over- expression of SERCA. We anticipate that the findings from these studies will identify promising targets for therapies to improve fistula maturation outcomes. PUBLIC HEALTH RELEVANCE: Arteriovenous fistulas provide the optimal access for hemodialysis in patients with end stage renal disease, yet nearly 50% of fistulas fail soon after their surgical construction due to venous smooth muscle hyperplasia that occludes the vascular lumen. At the time of AV fistula surgery, we will obtain fragments of vein for smooth muscle cell culture and immunohistochemistry. We will identify abnormalities in nitric oxide function, oxidant production, and intracellular calcium control that dictate smooth muscle cell proliferation and migration that may predict failure of the AV fistula, and therefore provide therapeutic targets to improve outcome.